CN114432285A

CN114432285A - Application of Resatorvid in up-regulating EFTUD2 expression and inhibiting HBV (hepatitis B virus) drugs

Info

Publication number: CN114432285A
Application number: CN202210213019.9A
Authority: CN
Inventors: 朱传龙; 蔡金原; 李毓雯; 刘瑞; 李军; 方慧晶; 李文庭
Original assignee: Jiangsu Province Hospital First Affiliated Hospital Of Nanjing Medical University
Current assignee: Jiangsu Province Hospital First Affiliated Hospital Of Nanjing Medical University
Priority date: 2022-03-06
Filing date: 2022-03-06
Publication date: 2022-05-06

Abstract

The invention provides the application of resatorvid in EFTUD2 up-regulation and in HBV inhibiting medicines. Wherein said resatorvid structural formula is as follows:

the compound can remarkably up-regulate EFTUD2 expression, can effectively inhibit HBV, has good inhibition effect on various indexes of HBV, particularly on HBV DNA, HBsAg, total HBV RNA, HBV 3.5-kb RNA and HBV cccDNA, can provide brand-new treatment options for hepatitis B patients with poor response of interferon treatment, and lays a foundation for developing new molecular targeted immunoregulation medicaments in the future.

Description

Application of Resatorvid in up-regulating EFTUD2 expression and inhibiting HBV (hepatitis B virus) drugs

Technical Field

The invention relates to the field of pharmacy, in particular to application of resatorvid in up-regulating EFTUD2 expression and inhibiting HBV (hepatitis B virus) medicines.

Background

China belongs to a moderate epidemic area of HBV infection, and about 2000 to 3000 ten thousand cases of the existing chronic hepatitis B patients are estimated. Although there are many drug choices for antiviral treatment of chronic hepatitis B, there are still many disadvantages, such as difficulty in clearing viral replication template cccDNA by nucleoside (nucleotide) analogues, low response rate of interferon, and large side effects. The optimization of the existing antiviral strategy still has specific value, but with the increasing demand of patients on 'functional cure', a plurality of scholars at home and abroad begin to explore new targets of immunotherapy and novel antiviral drugs in order to cure the disease.

Disclosure of Invention

In order to solve the above technical problems, the applicant has conducted intensive and long-term studies on inhibition of HBV and found that an intranuclear protein molecule EFTUD2 having a function of splicing pre-mRNA regulates the antiviral action of interferon. Clinical specimen detection shows that the liver tissue EFTUD2 baseline expression level of patients with HBeAg serological conversion in chronic hepatitis B interferon treatment is obviously higher than that of patients without response, and the EFTUD2 expression down-regulation can influence the anti-HBV capacity of interferon. In view of the above, the applicant imagines that the EFTUD2 is used as a target to perform high-throughput screening from a small molecule compound library, and the screened small molecule compound rescatorvid targeting the positive regulation EFTUD2 provides a novel treatment selection for hepatitis b patients, and lays a foundation for developing new molecular targeting immunomodulatory drugs.

To this end, applicants have conducted a dedicated study on resatorvid, a specific small molecule specific inhibitor of Toll-like receptor (TLR)4 signaling that interferes with the production of lipopolysaccharide-induced inflammatory mediators by binding to the intracellular domain of TLR 4. TLR4 can be activated by endogenous molecules released from damaged tissues or necrotic cells, and is also sensitive to exogenous molecules such as many bacteria that elicit the immune response of the host. Excessive activation of TLR4 is widely implicated in autoimmune/inflammatory diseases, including high mortality diseases such as sepsis, neurological related diseases, cardiovascular diseases, and many autoimmune diseases. In recent years, researchers have extensively studied the use of TLR4 inhibitors in various diseases, and the TLR4 inhibitors can be used as targets for treating diseases such as new coronary pneumonia, septicemia, liver failure and acute respiratory distress syndrome, and attract great attention.

However, inhibition of TLR4 may lead to a reduction in the production of many cytokines, such as tumor necrosis factor: (

) And interleukin-6 (IL-6), wherein IL-6 is reported to inhibit HBV replication in primary hepatocytes. In the present invention, the maximum working concentration of resatorvid is 0.5nm, which is not sufficient for

(IC 50 =1.9 nm) and IL-6 (IC 50 =1.3 nm) form significant inhibition, so the major pathway of resatorvid in the in vitro anti-HBV process is not through inhibition of TLR4, but through upregulation of EFTUD 2.

Based on multiple experiments, applicants found that resatorvid can up-regulate EFTUD2 expression in vitro and can inhibit HBsAg, total HBV RNA, 3.5-kbRNA, HBV DNA and HBV cccDNA levels. This finding suggests that a novel pharmaceutical use of resortorvid to up-regulate EFTUD2 and be effective against HBV may be uncovered.

To this end, the present invention proposes the use of resatorvid to up-regulate EFTUD2 expression and inhibit HBV;

wherein the structural formula of the resoratorvid is as follows:

further, the use of resatorvid for up-regulating EFTUD2 expression and inhibiting HBV drugs is proposed.

Further, a drug in which resatorvid up-regulates EFTUD2 expression and inhibits HBV is proposed.

Further, the expression of the EFTUD2 is up-regulated by EFTUD2 promoter activity, gene, protein expression and the like.

Further, the inhibition of HBV is the inhibition of HBsAg, total HBV RNA, 3.5-kb RNA, HBV DNA and HBV cccDNA levels.

Further, the use of resatorvid up-regulates EFTUD2 expression in vitro and can inhibit the levels of HBsAg, total HBV RNA, 3.5-kb RNA, HBV DNA and HBV cccDNA.

Further, drugs are proposed in which resatorvid up-regulates EFTUD2 expression in vitro, inhibiting HBsAg, total HBV RNA, 3.5-kb RNA, HBV DNA and HBV cccDNA levels.

Further, resatorvid up-regulates the expression of EFTUD2 in vitro, and can inhibit the use of HBsAg, total HBV RNA, 3.5-kb RNA, HBV DNA and HBV cccDNA level drugs.

Further, the HBV inhibition is an index of inhibiting HBV DNA, or HBsAg, or HBeAg, or HBV DNA, or total HBV RNA, or HBV 3.5-kb RNA, or HBV cccDNA and the like by using the resoratorvid alone or in combination.

Further, resatorvid up-regulates EFTUD2 expression in vitro or in vitro, and inhibits HBsAg, total HBV RNA, 3.5-kb RNA, HBV DNA and HBV cccDNA level.

Further, the use of resatorvid to up-regulate EFTUD2 drugs in HepAD38 and or HepG2-NTCP cells.

Further, resatorvid upregulates the activity of EFTUD2 in HepAD38 and or HepG2-NTCP cells.

Further, the use of resatorvid in anti-HBV drugs in HepAD38 cells. Further, a drug of resatorvid against HBV in HepAD38 cell was proposed.

Further, the resatorvid can inhibit HBV replication and transcription in an HBV in-vitro infected cell model, and can be used in a medicament for enhancing the anti-HBV effect after being used together with ETV. Further, resatorvid inhibits HBV replication and transcription in an HBV in vitro infected cell model, and enhances anti-HBV effects when used in combination with ETV.

Furthermore, the invention also provides an application of the resatorvid and ETV pharmaceutical composition in combination to improving the anti-HBV curative effect. Further, a pharmaceutical composition for improving the anti-HBV efficacy by combining resortorvid and ETV is provided.

The resatorvid compounds of the present invention are commercially available or can be prepared synthetically.

Has the advantages that: the compound rescatorvid has good up-regulation effect on EFTUD2, has good inhibition effect on various indexes of HBV, particularly on HBsAg, total HBV RNA, 3.5-kb RNA, HBV DNA and HBV cccDNA, and can provide a novel treatment selection for hepatitis B patients with poor response to interferon treatment. In one embodiment, resatorvid is used in combination with ETV to further improve the anti-HBV efficacy, with good drug application prospects.

Description of the drawings:

FIG. 1 inhibition curves of Resatorvid action on HepAD38 cells.

FIG. 2-inhibition curves of Resatorvid action on HepG2-NTCP cells.

FIG. 3 upregulation of EFTUD2 by Resatorvid in HepAD38 cells.

FIG. 4 upregulation of EFTUD2 by Resatorvid in HepG2-NTCP cells.

Figure 5 is a series of anti-HBV effects of Resatorvid in a HBV replicating cell model, wherein:

FIG. 5-1, Resatorvid inhibits the effect of supernatant HBV DNA in a HBV replicating cell model;

FIG. 5-2, Resatorvid inhibits the effect of supernatant HBsAg secretion in HBV replicating cell model;

FIGS. 5-3, Resatorvid inhibits intracellular HBV DNA effects in an HBV replicating cell model;

FIGS. 5-4, Resatorvid inhibits the effect of HBV total RNA in a HBV replicating cell model;

FIGS. 5-5, Resatorvid inhibits the effect of HBV 3.5-kb RNA in a HBV replicating cell model;

FIG. 5-6, Southern blot assay for full length genome of HBV DNA.

Figure 6 is a series of anti-HBV effects of Resatorvid in a model of HBV infected cells, wherein:

FIG. 6-1, Resatorvid inhibits the effect of supernatant HBV DNA in a cell model infected with HBV in vitro;

FIG. 6-2, Resatorvid inhibits the effect of supernatant HBsAg in a cell model infected with HBV in vitro;

FIGS. 6-3, Resatorvid inhibits intracellular HBV DNA effects in an in vitro HBV infected cell model;

FIGS. 6-4, Resatorvid inhibits the effect of HBV total RNA in a model of HBV infected cells in vitro;

FIGS. 6-5, Resatorvid inhibits the effect of HBV 3.5-kb RNA in a model of HBV infected cells in vitro;

6-6, Resatorvid can inhibit HBV cccDNA effects in HBV in vitro infected cell model;

FIG. 6-7, Southern blot assay for full length genome of HBV DNA.

The specific implementation mode is as follows:

the technical scheme of the invention is further described in the following with the accompanying drawings.

The following examples relate to primer sequences:

HBV DNA：

HBV DNA forward 5’-CCTAGTAGTCAGTTATGTCAAC-3’

HBV DNA reverse 5’-TCTATAAGCTGGAGGAGTGCGA-3’

total HBV RNA:

total HBV RNAs forward 5’- ACCGACCTTGAGGCATACTT-3’

total HBV RNAs reverse 5’- GCCTACAGCCTCCTAGTACA-3’

HBV 3.5-kb RNA：

HBV 3.5-kb RNA forward 5’- GCCTTAGAGTCTCCTGAGCA-3’

HBV 3.5-kb RNA reverse 5’- GAGGGAGTTCTTCTTCTAGG-3’

HBV cccDNA：

cccDNA forward 5’- CTCCCCGTCTGTGCCTTCT-3’

cccDNA reverse 5’-CCCCAAAGCCACCCAAG-3’

cccDNA probe 5’-TTCATCCTGCTGCTATGCCTGATCTTCTTG-3’

experiment of drug effect

(I) Main test materials

Resatorvid is available from MCE; fetal bovine serum was purchased from Gibco, usa; DMEM medium was purchased from Gibco, USA; Opti-MEM medium was purchased from Gibco, USA; PBS was purchased from sumo siemer feishell instruments ltd; D-Hank's Solution was purchased from Wuhan Punuoise Life technologies, Inc.; Trypsin-EDTA Solution was purchased from Shanghai Bin Yuntan corporation; L-Glutamine solution was purchased from Sigma-Aldrich, USA.

(II) cell culture

The following two cell lines were used in this study: human hepatoma cell lines HepAD38 and HepG 2-NTCP. The cells were cultured in DMEM medium containing 10% fetal bovine serum under conditions of 5% C02-95% air, saturated humidity and 37 ℃.

(III) MTT test

HepAD38 and HepG2-NTCP cells with good growth state are respectively inoculated into a 96-well plate at 5000/well, restatorvid with different concentrations is added, 3 multiple wells, a negative control well and a blank well are arranged, and the 96-well plate is placed in an incubator and treated with drugs for 48 hours. The plate was removed, 10. mu.l of MTT solution at a concentration of 5mg/ml was added to each well, and the incubation was continued in the incubator for 4 hours in the absence of light. Discard cell supernatant, add 100. mu.l of Formazan lysis solution to each well, and use blank wells to which 100. mu.l of Formazan lysis solution was added as zeroing wells. The plate was shaken on a shaker for 10 minutes to dissolve the formazan crystals completely, and the OD of each well was measured at a wavelength of 570nm in an ELISA analyzer.

Inhibition curves were fitted using GraphPad Prism 8 (GraphPad software) software with drug concentration as abscissa and corresponding cell activity as ordinate. The inhibition curves of Resatorvid on HepAD38 cells are shown in FIG. 1, and the inhibition curves on HepG2-NTCP cells are shown in FIG. 2.

(IV) detecting the upregulation of EFTUD2 by resatorvid

HepAD38 and HepG2-NTCP cells with good growth state are respectively cultured at 5X 10⁵Perwell was seeded in 6-well plates. The experimental groups were treated with resatorvid added to a final concentration of 0.5nM for 48 hours, setting 0.1% DMSO as a negative control.

Total intracellular RNA was extracted using the RNA-Quick Purification Kit (Yihua organism, Shanghai) and the purity and concentration of the product were checked using Nanodrop 2000 (Thermo Fisher Scientific, USA), A260/A280 was between 1.90-2.00 and the concentration (c) was adjusted to between 100 ng/μ l. The cDNA was produced by reverse transcription using a reverse transcription kit (RR 036A) from TARAKA. The expression of EFTUD2 was detected using GAPDH as an internal reference gene and a real-time fluorescent quantitative PCR kit (RR 820A) from TARAKA.

The results are shown in fig. 3 and 4, and 0.5nM resatorvid can significantly up-regulate the expression of EFTUD2 mRNA in HepAD38 and HepG2-NTCP cells, with good effect.

(V) testing anti-HBV effect of rescatorvid in HepAD38 cell

HepAD38 cells with good growth state were cultured at 5X 10⁵Perwell was seeded in 6-well plates. The experimental groups were added with resatorvid at final concentrations of 0.2nM, 0.5nM, and 1nM, respectively, and 0.1% DMSO was set as a negative control group and 25nM Entecavir (ETV) was set as a positive control group. HBV DNA in cell supernatant is detected respectively on

days

3, 6 and 9 (hepatitis B virus nucleic acid quantitative detection kit (fluorescent probe PCR method), Guangzhou Daan), HBsAg (hepatitis B virus surface antigen diagnostic kit (enzyme linked immunosorbent assay), Shanghai Kehua), and the operation method is carried out according to the kit instructions; detecting HBV DNA, HBV total RNA and HBV 3.5-kb RNA in cells by a PCR method; the Southern blot method detects the HBV DNA full-length genome.

The method for extracting the DNA in the cells comprises the following steps:

the cells were lysed with 0.5 ml of lysis buffer (10 mM Tris-HCl [ pH 8.0], 1 mM EDTA, 1% NP-40, 2% sucrose) at 37 ℃ for 15 minutes, and the cell debris and nuclei were removed by centrifugation. The supernatant was treated with DNase I at a final concentration of 40U/ml and 10 mM MgCl2 for 4 hours, then mixed with 200. mu.l of 35% PEG 8000 containing 1.5M NaCl and incubated on ice for 1 hour. Viral nucleocapsids were pelleted by centrifugation at 11000 Xg for 5 minutes at 4 ℃ and then digested overnight at 45 ℃ in 500. mu.l digestion buffer (containing 0.5 mg/ml proteinase K, 0.5% SDS, 150 mM NaCl, 25 mM Tris-HCl [ pH 8.0] and 10 mM EDTA). The digestion mixture was extracted twice with phenol, and DNA was precipitated with ethanol and dissolved in TE (10 mM Tris-HCl [ pH 8.0], 1 mM EDTA) buffer.

Southern blot experimental method:

the DNA samples extracted as described above were separated on a 0.9% agarose gel and purified. The DNA samples were transferred to nylon membranes overnight (Roche, Germany). After UV cross-linking and prehybridization, the membrane was spun and incubated with digoxin-labeled HBV-specific DNA probes generated by random primed DNA labeling kit (Roche, Germany) and the radiation signal was detected by exposure.

The rest methods are the same as the above. The ELISA results showed a significant reduction in HBsAg levels in the cell supernatant, specifically, at day 3/6/9, 0.1 nM resatorvid reduced HBsAg in the cell supernatant to 88%/83%/72% of the negative control, 79%/71%/63% in the 0.2nM group, and 73%/62%/50% in the 0.5nM group. In addition, PCR results also showed that resatorvid decreased intracellular and extracellular HBV DNA, intracellular HBV total RNA, 3.5-kb RNA levels in HepAD38 cells in a time-and dose-dependent manner. Finally, the full-length genome of HBV DNA was detected at about 3200bp by Southern blot experiment, and the result confirmed the significant decrease of HBV DNA. The above results indicate that resatorvid has inhibitory effect on HBV in the HBV replication model, as shown in the figure 5 series.

(VI) testing the anti-HBV effect of resatorvid in HBV in-vitro infected cell model

The method for constructing the HBV in-vitro infected cell model by using the HepG2-NTCP cells is researched previously, and specifically comprises the following steps: preparation of HBV inoculum: HepAD38 cells were grown to 80% confluence in medium containing doxycycline 2. mu.g/ml and G418 400. mu.g/ml, and cultured for another 10-14 days in medium without doxycycline and G418, and supernatants were collected every 2 days and stored at 4 ℃. All supernatants collected were centrifuged at 250 Xg for 20 min at 4 ℃ and the supernatants were taken and, after sterile filtration at 0.45 μm, PEG 8000 was added to a final concentration of 8%. Mix by repeated inversion overnight at 4 ℃. Centrifuging at 4 deg.C and 10000 Xg for 1 hr the next day, and removing supernatant to obtain purified HBV virus. The virus was resuspended in the appropriate DEME and the titer determined by PCR and stored at-80 ℃ until use. The medium was pretreated with doxycycline 2. mu.g/ml for 2-3 days to induce NTCP receptor expression.

In the HBV infection model, the experimental groups were: 0.5nM resatorvid treated group and 0.5nM resatorvid +25 nM ETV combination treated group, 0.1% DMSO was set as negative control group and 25nM Entecavir (ETV) was set as positive control group. HBV DNA, HBsAg in cell supernatant, HBV DNA, total HBV RNA, HBV 3.5-kb RNA, HBV cccDNA in cells were detected on day 10.

Method for extraction of cccDNA:

to selectively extract HBV cccDNA, infected HepG2-NTCP cells in the culture dish were taken, lysed with 1 ml lysis buffer at 37 ℃ for 60 min, added with 0.25 ml 2.5M KCl, and incubated overnight at 4 ℃ with 0.25 ml 2.5M KCl. The lysis buffer contained 50 mM Tris-HCl (pH = 7.4), 10 mM EDTA, 150 mM NaCl and 1% SDS, and did not contain proteinase K, and after centrifugation of the lysate at 12000g at 4 ℃ for 30 min, DNA was extracted with phenol and phenol-chloroform. The DNA was precipitated with an equal volume of isopropanol in the presence of 20. mu.g glycogen (Roche, Germany) and finally dissolved in TE buffer. The prepared DNA samples were then treated with plasmid-safe Adenosine Triphosphate (ATP) -dependent deoxyribonuclease (PSAD, Epicentre Technologies) according to the manufacturer's instructions. The rest methods are the same as the above.

PCR and ELISA results showed that resatorvid alone was also effective in inhibiting supernatant HBV DNA, HBsAg, intracellular HBV DNA, HBV total RNA, 3.5-kb RNA and HBV cccDNA levels in the HBV in vitro infected cell model; in addition, the combination of resatorvid + ETV has better antiviral effect by only reducing HBV DNA but not HBsAg and HBV RNA by ETV alone; finally, Southern blot analysis confirmed that resatorvid has antiviral effects in the HBV infection model. The results are shown in the series of FIG. 6.

Sequence listing

<110> Jiangsu province national hospital (the first subsidiary hospital of Nanjing medical university)

<120> use of Resatorvid for up-regulating EFTUD2 expression and inhibiting HBV

<160> 1

<170> SIPOSequenceListing 1.0

<210> 2

<211> 3182

<212> DNA

<213> Hepatitis B Virus (Hepatitis B virus)

<400> 2

aattccacaa cctttcacca aactctgcaa gatcccagag tgagaggcct gtatttccct 60

gctggtggct ccagttcagg agcagtaaac cctgttccga ctactgcctc tcccttatcg 120

tcaatcttct cgaggattgg ggaccctgcg ctgaacatgg agaacatcac atcaggattc 180

ctaggacccc ttctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata 240

ccgcagagtc tagactcgtg gtggacttct ctcaattttc tagggggaac taccgtgtgt 300

cttggccaaa attcgcagtc cccaacctcc aatcactcac caacctcctg tcctccaact 360

tgtcctggtt atcgctggat gtgtctgcgg cgttttatca tcttcctctt catcctgctg 420

ctatgcctca tcttcttgtt ggttcttctg gactatcaag gtatgttgcc cgtttgtcct 480

ctaattccag gatcctcaac caccagcacg ggaccatgcc gaacctgcat gactactgct 540

caaggaacct ctatgtatcc ctcctgttgc tgtaccaaac cttcggacgg aaattgcacc 600

tgtattccca tcccatcatc ctgggctttc ggaaaattcc tatgggagtg ggcctcagcc 660

cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc 720

actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacagcatc 780

ttgagtccct ttttaccgct gttaccaatt ttcttttgtc tttgggtata catttaaacc 840

ctaacaaaac aaagagatgg ggttactctc tgaattttat gggttatgtc attggaagtt 900

atgggtcctt gccacaagaa cacatcatac aaaaaatcaa agaatgtttt agaaaacttc 960

ctattaacag gcctattgat tggaaagtat gtcaacgaat tgtgggtctt ttgggttttg 1020

ctgccccatt tacacaatgt ggttatcctg cgttaatgcc cttgtatgca tgtattcaat 1080

ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatacctga 1140

acctttaccc cgttgcccgg caacggccag gtctgtgcca agtgtttgct gacgcaaccc 1200

ccactggctg gggcttggtc atgggccatc agcgcgtgcg tggaaccttt tcggctcctc 1260

tgccgatcca tactgcggaa ctcctagccg cttgttttgc tcgcagcagg tctggagcaa 1320

acattatcgg gactgataac tctgttgtcc tctcccgcaa atatacatcg tatccatggc 1380

tgctaggctg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg 1440

cgctgaatcc tgcggacgac ccttctcggg gtcgcttggg actctctcgt ccccttctcc 1500

gtctgccgtt ccgaccgacc acggggcgca cctctcttta cgcggactcc ccgtctgtgc 1560

cttctcatct gccggaccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac 1620

cgtgaacgcc caccgaatgt tgcccaaggt cttacataag aggactcttg gactctctgc 1680

aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaaag actgggagga 1740

gttgggggag gagattagat taaaggtctt tgtactagga ggctgtaggc ataaattggt 1800

ctgcgcacca gcaccatgca actttttcac ctctgcctaa tcatctcttg ttcatgtcct 1860

actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat cgacccttat 1920

aaagaatttg gagctactgt ggagttactc tcgtttttgc cttctgactt ctttccttca 1980

gtacgagatc ttctagatac cgcctcagct ctgtatcggg aagccttaga gtctcctgag 2040

cattgttcac ctcaccatac tgcactcagg caagcaattc tttgctgggg ggaactaatg 2100

actctagcta cctgggtggg tgttaatttg gaagatccag catctagaga cctagtagtc 2160

agttatgtca acactaatat gggcctaaag ttcaggcaac tcttgtggtt tcacatttct 2220

tgtctcactt ttggaagaga aaccgttata gagtatttgg tgtctttcgg agtgtggatt 2280

cgcactcctc cagcttatag accaccaaat gcccctatcc tatcaacact tccggaaact 2340

actgttgtta gacgacgagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga 2400

aggtctcaat cgccgcgtcg cagaagatct caatctcggg aacctcaatg ttagtattcc 2460

ttggactcat aaggtgggga actttactgg tctttattct tctactgtac ctgtctttaa 2520

tcctcattgg aaaacaccat cttttcctaa tatacattta caccaagaca ttatcaaaaa 2580

atgtgaacag tttgtaggcc cacttacagt taatgagaaa agaagattgc aattgattat 2640

gcctgctagg ttttatccaa aggttaccaa atatttacca ttggataagg gtattaaacc 2700

ttattatcca gaacatctag ttaatcatta cttccaaact agacactatt tacacactct 2760

atggaaggcg ggtatattat ataagagaga aacaacacat agcgcctcat tttgtgggtc 2820

accatattct tgggaacaag atctacagca tggggcagaa tctttccacc agcaatcctc 2880

tgggattctt tcccgaccac cagttggatc cagccttcag agcaaacaca gcaaatccag 2940

attgggactt caatcccaac aaggacacct ggccagacgc caacaaggta ggagctggag 3000

cattcgggct gggtttcacc ccaccgcacg gaggcctttt ggggtggagc cctcaggctc 3060

agggcatact acaaactttg ccagcaaatc cgcctcctgc ctccaccaat cgccagacag 3120

gaaggcagcc taccccgctg tctccacctt tgagaaacac tcatcctcag gccatgcagt 3180

gg 3182

Claims

Use of Resatorvid in up-regulating EFTUD2 expression and in inhibiting HBV drugs.
2. Use according to claim 1, characterized in that: the structural formula of the resatorvid is as follows:

use according to claim 1, characterized in that: the up-regulation of EFTUD2 expression is up-regulation of EFTUD2 promoter activity, gene, protein expression.
3. Use according to claim 1, characterized in that: the inhibition of HBV is the inhibition of HBsAg, total HBV RNA, 3.5-kb RNA, HBV DNA and HBV cccDNA level.
4. Use according to claim 1, characterized in that: the HBV inhibition is to inhibit HBV DNA, and/or HBsAg, and/or HBeAg, and/or HBV DNA, and/or total HBV RNA, and/or HBV 3.5-kb RNA, and/or HBV cccDNA by using resatorvid alone or in combination.
5. Use according to claim 1, characterized in that: resatorvid up-regulates EFTUD2 expression in vitro or in vitro, and inhibits HBsAg, and or total HBV RNA, and or 3.5-kb RNA, and or HBV DNA, and or HBV cccDNA.
6. Use according to claim 1, characterized in that: use of resatorvid to up-regulate EFTUD2 in HepAD38 and or HepG2-NTCP cells.
7. Use according to claim 1, characterized in that: use of resatorvid in an anti-HBV medicament in HepAD38 cells.
8. Use according to claim 1, characterized in that: resatorvid inhibits HBV replication and transcription in an HBV in vitro infected cell model, and enhances anti-HBV effect after being used together with ETV.
Use of Resatorvid in combination with an ETV drug to improve the efficacy of anti-HBV.